Chaperone-mediated autophagy (CMA) contributes to regulation of energy homeostasis by timely degradation of enzymes involved in glucose and lipid metabolism. Here, we report reduced CMA activity in... Show moreChaperone-mediated autophagy (CMA) contributes to regulation of energy homeostasis by timely degradation of enzymes involved in glucose and lipid metabolism. Here, we report reduced CMA activity in vascular smooth muscle cells and macrophages in murine and human arteries in response to atherosclerotic challenges. We show that in vivo genetic blockage of CMA worsens atherosclerotic pathology through both systemic and cell-autonomous changes in vascular smooth muscle cells and macrophages, the two main cell types involved in atherogenesis. CMA deficiency promotes dedifferentiation of vascular smooth muscle cells and a proinflammatory state in macrophages. Conversely, a genetic mouse model with up-regulated CMA shows lower vulnerability to proatherosclerotic challenges. We propose that CMA could be an attractive therapeutic target against cardiovascular diseases. Show less
In modern society, the widespread use of artificial light at night disrupts the suprachiasmatic nucleus (SCN), which serves as our central circadian clock. Existing models describe excitatory... Show moreIn modern society, the widespread use of artificial light at night disrupts the suprachiasmatic nucleus (SCN), which serves as our central circadian clock. Existing models describe excitatory responses of the SCN to primarily blue light, but direct measures in humans are absent. The combination of state-of-the-art neuroimaging techniques and custom-made MRI compatible light-emitting diode devices allowed to directly measure the light response of the SCN. In contrast to the general expectation, we found that blood oxygen level-dependent (BOLD) functional MRI signals in the SCN were suppressed by light. The suppressions were observed not only in response to narrowband blue light (lambda max: 470 nm) but remarkably, also in response to green (lambda max: 515 nm) and orange (lambda max: 590 nm), but not to violet light (lambda max: 405 nm). The broadband sensitivity of the SCN implies that strategies on light exposure should be revised: enhancement of light levels during daytime is possible with wavelengths other than blue, while during nighttime, all colors are potentially disruptive. Show less
Weigert Muñoz, A.; Hoyer, E.; Schumacher, K.; Grognot, M.; Taute, K.M.; Hacker, S.M.; ... ; Jung, K. 2022
In addition to their well-known role as stress-associated catecholamine hormones in animals and humans, epinephrine (EPI) and norepinephrine (NE) act as interkingdom signals between eukaryotic... Show moreIn addition to their well-known role as stress-associated catecholamine hormones in animals and humans, epinephrine (EPI) and norepinephrine (NE) act as interkingdom signals between eukaryotic hosts and bacteria. However, the molecular basis of their effects on bacteria is not well understood. In initial phenotypic studies utilizing Vibrio campbellii as a model organism, we characterized the bipartite mode of action of catecholamines, which consists of promotion of growth under iron limitation and enhanced colony expansion on soft agar. In order to identify the molecular targets of the hormones, we designed and synthesized tailored probes for chemical proteomic studies. As the catechol group in EPI and NE acts as an iron chelator and is prone to form a reactive quinone moiety, we devised a photoprobe based on the adrenergic agonist phenylephrine (PE), which solely influenced colony expansion. Using this probe, we identified CheW, located at the core of the chemotaxis signaling network, as a major target. In vitro studies confirmed that EPI, NE, PE, and labetalol, a clinically applied antagonist, bind to purified CheW with affinity constants in the submicromolar range. In line with these findings, exposure of V. campbellii to these adrenergic agonists affects the chemotactic control of the bacterium. This study highlights an effect of eukaryotic signaling molecules on bacterial motility. Show less
Insular woodiness (IW)—the evolutionary transition from herbaceousness toward wood- iness on islands—is one of the most iconic features of island floras. Since pioneering work by Darwin and Wallace... Show moreInsular woodiness (IW)—the evolutionary transition from herbaceousness toward wood- iness on islands—is one of the most iconic features of island floras. Since pioneering work by Darwin and Wallace, a number of drivers of IW have been proposed, such as 1) competition for sunlight requiring plants with taller and stronger woody stems and 2) drought favoring woodiness to safeguard root-to-shoot water transport. Alternatively, IW may be the indirect result of increased lifespan related to 3) a favorable aseasonal climate and/or 4) a lack of large native herbivores. However, information on the occurrence of IW is fragmented, hampering tests of these potential drivers. Here, we identify 1,097 insular woody species on 375 islands and infer at least 175 evolutionary transitions on 31 archipelagos, concentrated in six angiosperm families. Structural equation models reveal that the insular woody species richness on oceanic islands correlates with a favorable aseasonal climate, followed by increased drought and island isolation (approximating competition). When continental islands are also included, reduced herbivory pressure by large native mammals, increased drought, and island isolation are most relevant. Our results illustrate different trajectories leading to rampant convergent evolution toward IW and further emphasize archipelagos as natural laboratories of evolution, where similar abiotic or biotic conditions replicated evolution of similar traits. Show less
Hannikainen, I.R.; Tobia, K.P.; Almeida, G.d.F.C.F. de; Struchiner, N.; Kneer, M.; Bystranowski, P.; ... ; Żuradzki, T. 2022
As coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from... Show moreAs coronaviruses (CoVs) replicate in the host cell cytoplasm, they rely on their own capping machinery to ensure the efficient translation of their messenger RNAs (mRNAs), protect them from degradation by cellular 5 ' exoribonucleases (ExoNs), and escape innate immune sensing. The CoV nonstructural protein 14 (nsp14) is a bifunctional replicase subunit harboring an N-terminal 3 '-to-5 ' ExoN domain and a C-terminal (N7-guanine)-methyltransferase (N7-MTase) domain that is presumably involved in viral mRNA capping. Here, we aimed to integrate structural, biochemical, and virological data to assess the importance of conserved N7-MTase residues for nsp14's enzymatic activities and virus viability. We revisited the crystal structure of severe acute respiratory syndrome (SARS)-CoV nsp14 to perform an in silico comparative analysis between beta-coronaviruses. We identified several residues likely involved in the formation of the N7-MTase catalytic pocket, which presents a fold distinct from the Rossmann fold observed in most known MTases. Next, for SARS-CoV andMiddle East respiratory syndrome CoV, site-directed mutagenesis of selected residues was used to assess their importance for in vitro enzymatic activity. Most of the engineered mutations abolished N7-MTase activity, while not affecting nsp14-ExoN activity. Upon reverse engineering of these mutations into different betacoronavirus genomes, we identified two substitutions (R310A and F426A in SARS-CoV nsp14) abrogating virus viability and one mutation (H424A) yielding a crippled phenotype across all viruses tested. Our results identify the N7-MTase as a critical enzyme for betacoronavirus replication and define key residues of its catalytic pocket that can be targeted to design inhibitors with a potential pan-coronaviral activity spectrum. Show less
The voltage-gated sodium channel Nav1.5 initiates the cardiac action potential. Alterations of its activation and inactivation properties due to mutations can cause severe, life-threatening... Show moreThe voltage-gated sodium channel Nav1.5 initiates the cardiac action potential. Alterations of its activation and inactivation properties due to mutations can cause severe, life-threatening arrhythmias. Yet despite intensive research efforts, many functional aspects of this cardiac channel remain poorly understood. For instance, Nav1.5 undergoes extensive posttranslational modification in vivo, but the functional significance of these modifications is largely unexplored, especially under pathological conditions. This is because most conventional approaches are unable to insert metabolically stable posttranslational modification mimics, thus preventing a precise elucidation of the contribution by these modifications to channel function. Here, we overcome this limitation by using protein semisynthesis of Nav1.5 in live cells and carry out complementary molecular dynamics simulations. We introduce metabolically stable phosphorylation mimics on both wild-type (WT) and two pathogenic long-QT mutant channel backgrounds and decipher functional and pharmacological effects with unique precision. We elucidate the mechanism by which phosphorylation of Y1495 impairs steady-state inactivation in WT Nav1.5. Surprisingly, we find that while the Q1476R patient mutation does not affect inactivation on its own, it enhances the impairment of steady-state inactivation caused by phosphorylation of Y1495 through enhanced unbinding of the inactivation particle. We also show that both phosphorylation and patient mutations can impact Nav1.5 sensitivity toward the clinically used antiarrhythmic drugs quinidine and ranolazine, but not flecainide. The data highlight that functional effects of Nav1.5 phosphorylation can be dramatically amplified by patient mutations. Our work is thus likely to have implications for the interpretation of mutational phenotypes and the design of future drug regimens. Show less
Biosynthesis of sterols, which are key constituents of canonical eukaryotic membranes, requires molecular oxygen. Anaerobic protists and deep-branching anaerobic fungi are the only eukaryotes in... Show moreBiosynthesis of sterols, which are key constituents of canonical eukaryotic membranes, requires molecular oxygen. Anaerobic protists and deep-branching anaerobic fungi are the only eukaryotes in which a mechanism for sterol-independent growth has been elucidated. In these organisms, tetrahymanol, formed through oxygen-independent cyclization of squalene by a squalene-tetrahymanol cyclase, acts as a sterol surrogate. This study confirms an early report [C. J. E. A. Bulder, Antonie Van Leeuwenhoek, 37, 353-358 (1971)] that Schizosaccharomyces japonicus is exceptional among yeasts in growing anaerobically on synthetic media lacking sterols and unsaturated fatty acids. Mass spectrometry of lipid fractions of anaerobically grown Sch. japonicus showed the presence of hopanoids, a class of cyclic triterpenoids not previously detected in yeasts, including hop-22(29)-ene, hop17(21)-ene, hop-21(22)-ene, and hopan-22-ol. A putative gene in Sch. japonicus showed high similarity to bacterial squalene-hopene cyclase (SHC) genes and in particular to those of Acetobacter species. No orthologs of the putative Sch. japonicus SHC were found in other yeast species. Expression of the Sch. japonicus SHC gene (Sjshc1) in Saccharomyces cerevisiae enabled hopanoid synthesis and stimulated anaerobic growth in sterol-free media, thus indicating that one or more of the hopanoids produced by SjShc1 could at least partially replace sterols. Use of hopanoids as sterol surrogates represents a previously unknown adaptation of eukaryotic cells to anaerobic growth. The fast anaerobic growth of Sch. japonicus in sterol-free media is an interesting trait for developing robust fungal cell factories for application in anaerobic industrial processes. Show less
Embgenbroich, M.; Zande, H.J.P. van der; Hussaarts, L.; Schulte-Schrepping, J.; Pelgrom, L.R.; Garcia-Tardon, N.; ... ; Burgdorf, S. 2021
Proinflammatory activation of macrophages in metabolic tissues is critically important in the induction of obesity-induced metaflammation. Here, we demonstrate that the soluble mannose receptor ... Show moreProinflammatory activation of macrophages in metabolic tissues is critically important in the induction of obesity-induced metaflammation. Here, we demonstrate that the soluble mannose receptor (sMR) plays a direct functional role in both macrophage activation and metaflammation. We show that sMR binds CD45 on macrophages and inhibits its phosphatase activity, leading to an Src/Akt/ NF-kappa B-mediated cellular reprogramming toward an inflammatory phenotype both in vitro and in vivo. Remarkably, increased serum sMR levels were observed in obese mice and humans and directly correlated with body weight. Importantly, enhanced sMR levels increase serum proinflammatory cytokines, activate tissue macrophages, and promote insulin resistance. Altogether, our results reveal sMR as regulator of proinflammatory macrophage activation, which could constitute a therapeutic target for metaflammation and other hyperinflammatory diseases. Show less
Complement is an important effector mechanism for antibodymediated clearance of infections and tumor cells. Upon binding to target cells, the antibody's constant (Fc) domain recruits complement... Show moreComplement is an important effector mechanism for antibodymediated clearance of infections and tumor cells. Upon binding to target cells, the antibody's constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr2s2) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r2s2). While interactions between C1 and IgG-Fc are believed to be mediated by the globular heads of C1q, we here find that C1r2s2 proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various 2,4-dinitrophenol [DNP]-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r2s2 contributes to C1q-IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG, we reveal that hexamer-enhancing mutations improve C1q-IgG stability, both in the absence and presence of C1r2s2. In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies. Show less
Ambient light detection is important for the synchronization of the circadian clock to the external solar cycle. Light signals are sent to the suprachiasmatic nuclei (SCN), the site of the major... Show moreAmbient light detection is important for the synchronization of the circadian clock to the external solar cycle. Light signals are sent to the suprachiasmatic nuclei (SCN), the site of the major circadian pacemaker. It has been assumed that cone photoreceptors con-tribute minimally to synchronization. Here, however, we find that cone photoreceptors are sufficient for mediating entrainment and transmitting photic information to the SCN, as evaluated in mice that have only cones as functional photoreceptors. Using in vivo electrophysiological recordings in the SCN of freely moving cone-only mice, we observed light responses in SCN neuronal activity in response to 60-s pulses of both ultraviolet (UV) (lambda(max) 365 nm) and green (lambda(max) 505 nm) light. Higher irradiances of UV light led to irradiance-dependent enhancements in SCN neuronal activity, whereas higher irradiances of green light led to a reduction in the sustained response with only the transient response remain-ing. Responses in SCN neuronal activity decayed with a half-max time of similar to 9 min for UV light and less than a minute for green light, indicating differential input between short-wavelength-sensitive and mid-wavelength-sensitive cones for the SCN responsiveness. Furthermore, we show that UV light is more effective for photo-entrainment than green light. Based on the lack of a full sustained response in cone-only mice, we confirmed that rapidly alternating light levels, rather than slowly alternating light, caused substantial phase shifts. Together, our data provide strong evidence that cone types contribute to photoentrainment and differentially affect the electrical activity levels of the SCN. Show less
Human immunoglobulin (Ig) G4 usually displays antiinflammatory activity, and observations of IgG4 autoantibodies causing severe autoimmune disorders are therefore poorly understood. In blood, IgG4... Show moreHuman immunoglobulin (Ig) G4 usually displays antiinflammatory activity, and observations of IgG4 autoantibodies causing severe autoimmune disorders are therefore poorly understood. In blood, IgG4 naturally engages in a stochastic process termed "Fab-arm exchange" in which unrelated IgG4s exchange half-molecules continuously. The resulting IgG4 antibodies are composed of two different binding sites, thereby acquiring monovalent binding and inability to cross-link for each antigen recognized. Here, we demonstrate that this process amplifies autoantibody pathogenicity in a classic IgG4-mediated autoimmune disease: muscle-specific kinase (MuSK) myasthenia gravis. In mice, monovalent anti-MuSK IgG4s caused rapid and severemyasthenicmuscleweakness, whereas the same antibodies in their parental bivalent form were less potent or did not induce a phenotype. Mechanistically this could be explained by opposing effects onMuSK signaling. Isotype switching to IgG4 in an autoimmune response thereby may be a critical step in the development of disease. Our study establishes functional monovalency as a pathogenic mechanism in IgG4-mediated autoimmune disease and potentially other disorders. Show less
Endogenous mediators regulating acute inflammatory responses in both the induction and resolution phases of inflammatory processes are pivotal in host defense and tissue homeostasis. Recent studies... Show moreEndogenous mediators regulating acute inflammatory responses in both the induction and resolution phases of inflammatory processes are pivotal in host defense and tissue homeostasis. Recent studies have identified neuronal guidance proteins characterized in axonal development that display immunomodulatory functions. Here, we identify the neuroimmune guidance cue Semaphorin 7A (Sema7A), which appears to link macrophage (M Phi) metabolic remodeling to inflammation resolution. Sema7A orchestrated M Phi chemotaxis and chemokinesis, activated M Phi differentiation and polarization toward the proresolving M2 phenotype, and promoted leukocyte clearance. Peritoneal M Phi(sema7A-/-) displayed metabolic reprogramming, characterized by reductions in fatty acid oxidation and oxidative phosphorylation, increases in glycolysis and the pentose phosphate pathway, and truncation of the tricarboxylic acid cycle, which resulted in increased levels of the intermediates succinate and fumarate. The low accumulation of citrate in M Phi(sema7A-/-) correlated with the decreased synthesis of prostaglandins, leading to a reduced impact on lipid-mediator class switching and the generation of specialized pro resolving lipid mediators. Signaling network analysis indicated that Sema7A induced the metabolic reprogramming of M Phi by activating the mTOR-and AKT2-signaling pathways. Administration of Sema7A(SL4cd) orchestrated the resolution response to tissue homeostasis by shortening the resolution interval, promoting tissue protection in murine peritonitis, and enhancing survival in polymicrobial sepsis. Show less
RNA-dependent RNA polymerases (RdRps) of the Nidovirales (Coronaviridae, Arteriviridae, and 12 other families) are linked to an amino-terminal (N-terminal) domain, called NiRAN, in a non-structural... Show moreRNA-dependent RNA polymerases (RdRps) of the Nidovirales (Coronaviridae, Arteriviridae, and 12 other families) are linked to an amino-terminal (N-terminal) domain, called NiRAN, in a non-structural protein (nsp) that is released from polyprotein 1ab by the viral main protease (Mpro). Previously, self-GMPylation/UMPylation activities were reported for an arterivirus NiRAN-RdRp nsp and suggested to generate a transient state primed for transferring nucleoside monophosphate (NMP) to (currently unknown) viral and/or cellular biopolymers. Here, we show that the coronavirus (human coronavirus [HCoV]-229E and severe acute respiratory syndrome coronavirus 2) nsp12 (NiRAN-RdRp) has Mn2+-dependent NMPylation activity that catalyzes the transfer of a single NMP to the cognate nsp9 by forming a phosphoramidate bond with the primary amine at the nsp9 N terminus (N3825) following M-pro-mediated proteolytic release of nsp9 from N-terminally flanking nsps. Uridine triphosphate was the preferred nucleotide in this reaction, but also adenosine triphosphate, guanosine triphosphate, and cytidine triphosphate were suitable cosubstrates. Mutational studies using recombinant coronavirus nsp9 and nsp12 proteins and genetically engineered HCoV-229E mutants identified residues essential for NiRAN-mediated nsp9 NMPylation and virus replication in cell culture. The data corroborate predictions on NiRAN active-site residues and establish an essential role for the nsp9 N3826 residue in both nsp9 NMPylation in vitro and virus replication. This residue is part of a conserved N-terminal NNE tripeptide sequence and shown to be the only invariant residue in nsp9 and its homologs in viruses of the family Coronaviridae. The study provides a solid basis for functional studies of other nidovirus NMPylation activities and suggests a possible target for antiviral drug development. Show less
Wenzel, M.; Gulsoy, I.N.C.; Gao, Y.Q.; Teng, Z.H.; Willemse, J.J.; Middelkamp, M.; ... ; Hamoen, L.W. 2021
Gram-positive bacteria divide by forming a thick cross wall. How the thickness of this septal wall is controlled is unknown. In this type of bacteria, the key cell division protein FtsZ is anchored... Show moreGram-positive bacteria divide by forming a thick cross wall. How the thickness of this septal wall is controlled is unknown. In this type of bacteria, the key cell division protein FtsZ is anchored to the cell membrane by two proteins, FtsA and/or SepF. We have isolated SepF homologs from different bacterial species and found that they all polymerize into large protein rings with diameters varying from 19 to 44 nm. Interestingly, these values correlated well with the thickness of their septa. To test whether ring diameter determines septal thickness, we tried to construct different SepF chimeras with the purpose to manipulate the diameter of the SepF protein ring. This was indeed possible and confirmed that the conserved core domain of SepF regulates ring diameter. Importantly, when SepF chimeras with different diameters were expressed in the bacterial host Bacillus subtilis, the thickness of its septa changed accordingly. These results strongly support a model in which septal thickness is controlled by curved molecular clamps formed by SepF polymers attached to the leading edge of nascent septa. This also implies that the intrinsic shape of a protein polymer can function as a mold to shape the cell wall. Show less
Barone, M.; Muller, M.; Chiha, S.; Ren, J.; Albat, D.; Soicke, A.; ... ; Kuhne, R. 2020
Battling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular... Show moreBattling metastasis through inhibition of cell motility is considered a promising approach to support cancer therapies. In this context, Ena/VASP-depending signaling pathways, in particular interactions with their EVH1 domains, are promising targets for pharmaceutical intervention. However, protein-protein interactions involving proline-rich segments are notoriously difficult to address by small molecules. Hence, structure-based design efforts in combination with the chemical synthesis of additional molecular entities are required. Building on a previously developed nonpeptidic micromolar inhibitor, we determined 22 crystal structures of ENAH EVH1 in complex with inhibitors and rationally extended our library of conformationally defined prolinederived modules (ProMs) to succeed in developing a nanomolar inhibitor (K-d = 120 nM, MW = 734 Da). In contrast to the previous inhibitor, the optimized compounds reduced extravasation of invasive breast cancer cells in a zebrafish model. This study represents an example of successful, structure-guided development of low molecular weight inhibitors specifically and selectively addressing a proline-rich sequence-recognizing domain that is characterized by a shallow epitope lacking defined binding pockets. The evolved high-affinity inhibitor may now serve as a tool in validating the basic therapeutic concept, i.e., the sup pression of cancer metastasis by inhibiting a crucial protein- protein interaction involved in actin filament processing and cell migration. Show less
Kwesi-Maliepaard, E.M.; Aslam, M.A.; Alemdehy, M.F.; Brand, T. van den; McLean, C.; Vlaming, H.; ... ; Jacobs, H. 2020
Cytotoxic T cell differentiation is guided by epigenome adaptations, but how epigenetic mechanisms control lymphocyte development has not been well defined. Here we show that the histone... Show moreCytotoxic T cell differentiation is guided by epigenome adaptations, but how epigenetic mechanisms control lymphocyte development has not been well defined. Here we show that the histone methyltransferase DOT1L, which marks the nucleosome core on active genes, safeguards normal differentiation of CD8(+) T cells. T cell-specific ablation of DOT1L resulted in loss of naive CD8(+) T cells and premature differentiation toward a memory-like state, independent of antigen exposure and in a cell-intrinsic manner. Mechanistically, DOT1L controlled CD8(+) T cell differentiation by ensuring normal T cell receptor density and signaling. DOT1L also maintained epigenetic identity, in part by indirectly supporting the repression of developmentally regulated genes. Finally, deletion of DOT1L in T cells resulted in an impaired immune response. Through our study, DOT1L is emerging as a central player in physiology of CD8(+) T cells, acting as a barrier to prevent premature differentiation and controlling epigenetic integrity. Show less
Hsu, C.C.; Xu, J.B.; Brinkhof, B.; Wang, H.; Cui, Z.F.; Huang, W.E.; Ye, H. 2020
Stem cells with the capability to self-renew and differentiate into multiple cell derivatives provide platforms for drug screening and promising treatment options for a wide variety of neural... Show moreStem cells with the capability to self-renew and differentiate into multiple cell derivatives provide platforms for drug screening and promising treatment options for a wide variety of neural diseases. Nevertheless, clinical applications of stem cells have been hindered partly owing to a lack of standardized techniques to characterize cell molecular profiles noninvasively and comprehensively. Here, we demonstrate that a label-free and noninvasive single-cell Raman microspectroscopy (SCRM) platform was able to identify neural cell lineages derived from clinically relevant human induced pluripotent stem cells (hiPSCs). By analyzing the intrinsic biochemical profiles of single cells at a large scale (8,774 Raman spectra in total), iPSCs and iPSC-derived neural cells can be distinguished by their intrinsic phenotypic Raman spectra. We identified a Raman biomarker from glycogen to distinguish iPSCs from their neural derivatives, and the result was verified by the conventional glycogen detection assays. Further analysis with a machine learning classification model, utilizing t-distributed stochastic neighbor embedding (t-SNE)-enhanced ensemble stacking, clearly categorized hiPSCs in different develop- mental stages with 97.5% accuracy. The present study demon-strates the capability of the SCRM-based platform to monitor cell development using high content screening with a noninvasive and label-free approach. This platform as well as our identified bio- marker could be extensible to other cell types and can potentially have a high impact on neural stem cell therapy. Show less
Garcia-Rodriguez, R.; Hiller, M.; Jimenez-Gracia, L.; Pal, Z. van der; Balog, J.; Adamzek, K.; ... ; Spitali, P. 2020
Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene leading to the presence of premature termination codons (PTC). Previous transcriptional studies have shown reduced DMD... Show moreDuchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene leading to the presence of premature termination codons (PTC). Previous transcriptional studies have shown reduced DMD transcript levels in DMD patient and animal model muscles when PTC are present. Nonsense-mediated decay (NMD) has been suggested to be responsible for the observed reduction, but there is no experimental evidence supporting this claim. In this study, we aimed to investigate the mechanism responsible for the drop in DMD expression levels in the presence of PTC. We observed that the inhibition of NMD does not normalize DMD gene expression in DMD. Additionally, in situ hybridization showed that DMD mes-senger RNA primarily localizes in the nuclear compartment, con-firming that a cytoplasmic mechanism like NMD indeed cannot be responsible for the observed reduction. Sequencing of nascent RNA to explore DMD transcription dynamics revealed a lower rate of DMD transcription in patient-derived myotubes compared to healthy controls, suggesting a transcriptional mechanism involved in reduced DMD transcript levels. Chromatin immunoprecipitation in muscle showed increased levels of the repressive histone mark H3K9me3 in mdx mice compared to wild-type mice, indicating a chromatin conformation less prone to transcription in mdx mice. In line with this finding, treatment with the histone deacetylase in-hibitor givinostat caused a significant increase in DMD transcript expression in mdx mice. Overall, our findings show that transcrip-tion dynamics across the DMD locus are affected by the presence of PTC, hinting at a possible epigenetic mechanism responsible for this process. Show less
